Botulinum neurotoxins (BoNTs) are the most toxic substances known to man, and cause the deadly neuroparalytic disease, botulism. The flaccid paralysis caused by botulism lasts for months. BoNTs can be used as potential bioterrorism agents, and therefore, pose great threat to homeland security and public health. Currently no effective and safe antidote exists for prophylactic and therapeutic treatment for botulism. The only approved antidotes are equine antitoxins, which can only neutralize the toxin in the blood circulation, and have a very short treatment window. In addition, the equine antitoxins can cause severe side effects, excluding them as effective prophylactics. The only alternative for patients remains to be put on artificial ventilation under intensive care for several months. In this R21 exploratory research grant, we propose a new generation of antidotes against botulism, aptamers. Aptamers are unique oligonucleotides that have high affinity for their targets, ranging from small molecules to proteins. Aptamers have low toxicity, no reported immunogenicity, low cost to manufacture, and excellent storage stability. Those characteristics make aptamers ideal as antidotes against botulism. The goal of this research is to develop aptamers as antidotes against botulism. The proposed antidotes have potential to be both effective prophylactic treatment, and effective therapeutic drugs to treat botulism patients with symptoms. The specific aims to be pursued in the next two years are: (1) Selection and characterization of aptamers against BoNT. We will use SELEX to screen random RNA libraries, isolate the aptamers against BoNT, and prioritize aptamers based on binding affinity to BoNT. (2) Validation of anti-BoNT activity of aptamers. We will determine the inhibitory effects on both endopeptidase activity and toxin binding to its receptors, using both in vitro and cell-based assays. The aptamers identified from this study, therefore, will be used as lead compounds for the designing of more potent antidotes against botulism. This study will lead to a new class of antidotes against botulism. The research approach can be readily applicable to all seven serotypes of BoNT and other priority pathogens on biodefense agenda. In addition, the research proposed here will have significant impact on the rapid diagnostics of biodefense agents. The ever increasing health risks due to bioterrorism worldwide have placed a large burden on the scientific community to rapidly develop safe prophylactic and therapeutic treatments for biological and chemical toxins. This task has been a high priority for public health. There is lack of therapeutic and prophylactic drugs for botulism and most of the other biological toxins (as listed in NIAID priority pathogen list). Our project will result in aptamer-based drug candidates against botulism, a new class of antidotes against botulism, with many advantages over currently available antitoxin.